Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Site-selective protein–protein
coupling has long been a
goal of chemical biology research. In recent years, that goal has
been realized to varying degrees through a number of techniques, including
the use of tyrosinase-based coupling strategies. Early publications
utilizing tyrosinase from Agaricus bisporus­(abTYR) showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging
to produce recombinantly and suffers from some limitations in substrate
scope. Initial screens of several tyrosinase candidates revealed that
the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme that possesses a broad substrate
tolerance. We use the expanded substrate preference as a starting
point for protein design experiments and show that single point mutants
of megaTYR are capable of activating tyrosine residues in various
sequence contexts. We leverage this new tool to enable the construction
of protein trimers via a charge-directed sequential activation of
tyrosine residues (CDSAT).